Toward a circular bioeconomy: extracting cellulose from grape

: T he purpose of this study was to assess the extraction of cellulose from stalks of vines


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In recent times, there has been an increase in the demand for cellulose fibers, where 24 the main source is wood pulp, however, due to the depletion of natural resources and 25 global warming, several kinds of research are being developed to replace this scarce re-26 source, as alternative sources such as vegetable residues from agricultural activities and 27 agro-industries are being sought to avoid deforestation [1,2]. Among these residues, high-28 lighted in this study is the grape stem, which is obtained after destemming. The grape 29 stem can represent up to 5 − 7 % of the raw material used in processing, are the skeletons 30 of the grape bunch and are composed mainly of lignocelluloses [3,4]. According to recent 31 studies, grape stems are intrinsically dangerous; however, they have a high content of 32 organic matter and the fact that production is concentrated at one time of the year presents 33 potential pollution problems [4,5], that is, the discharge of grape stems into the soil leads 34 to the inhibition of the germinative properties of the soil, due to the biological demand of 35 oxygen, carbon and phenolic compounds [4,6]. Thus, to obtain this polymer of great im-36 portance from the grape stem, there are numerous methods of extraction among them 37 acid hydrolysis and autohydrolysis are the most used approaches to the extraction of cel-38 lulose. The most commonly used acids are acetic acid, sulfuric acid, and phosphoric acid, 39 for acid hydrolysis [7]. Factors to be considered as advantages of acid hydrolysis are the 40 low release of lignin fragments and the efficient hydrolysis of amorphous polysaccha-41 rides. However, the high cost of acid recovery and the corrosion of equipment are consid-42 ered the disadvantages of acid hydrolysis [8]. Autohydrolysis is acidic hydrolysis without 43 the use of any external acid. Because hydrogen ions come from the autoionization of water 44 in situ and acetic acid generated from the acetyl substituent of hemicellulose. The latter 45 contributes much more to hydrolysis [8][9][10]. The acid-base method is the most used 1 method for cellulose extraction, its advantages include a simple extraction process, high 2 extraction efficiency, good thermal stability, good crystallinity, easy control of reaction 3 conditions and low cost. The beneficial thing about this method of pulp extraction is to 4 achieve large-scale industrial production and has ample development space [11]. There-5 fore, in the present study, the objective was to evaluate the extraction, by means of acid 6 hydrolysis, alkaline hydrolysis and bleaching of cellulose from residues of the wine in-7 dustry, focusing on the valorization of the grape stem ('Vinhão') and to characterize the 8 effects of granulometric fractions and Retain (500, 300, 250, 150, 150) μm in relation to 9 color, FTIR and light microscopy. The final application of the cellulose will be the creation 10 of membranes/coatings through a sustainable and environmentally friendly process for 11 the protection of heritage buildings from environmental conditions. Grape stalks from ´Vinhao` variety were kindly provided by Quinta de Mascate (Vila 15 Verde, Portugal). The vine stalks were collected from (meter local) and frozen until used. 16 The samples were dried at 60 •C for 48 hours in an oven with air circulation. The dried 17 biomass was then micronized with a bimby (TM5) and sieved for grain-size classification 18 within the range from 500 µm to 150 µm (500, 300, 250, 150) µm and retain Fig 1. The milled 19 by-products were packed in sealed plastic bags, protected from light until use. To better 20 understand the extraction and bleaching of the by-products all fractions were used in this 21 study. 22 23 Cellulose was extracted based on the work of Bassani et al., [12], using the process of 24 auto-hydrolysis, with some modifications. The dried micronized by-product was 25 weighted 20 g and proceeded to an acid hydrolysis, carried out in a Schott with a ratio of 26 1 : 20 (m/V) of 5 % acid sulfuric, in autoclave (121 •C, 45 min ate 1.2 atm). The samples 27 were cooled at room temperature and washed with distilled water until waster become 28 clear. The samples were dried at 50 •C for 24 h. 29 The samples were submitted to 4 mol/L NaOH at a ratio 1 : 20 (m/V) and put into 30 orbital shaker at room temperature with 180 rpm for 24 h. The samples were cooled at 31 room temperature and washed with distilled water until waster become clear. The sam-32 ples were dried at 50 ºC for 24 h. 33 Lastly, the bleaching process was carried out by adding 1 : 20 (m/V) of 5 % H2O2 (the 34 pH was adjusted to 11.5 with NaOH), in an orbital shaker for 8 h at 45 •C. The sample 35 were taken out of the orbital shaker and cooled for 24 h at room temperature. The samples 36 were cooled at room temperature and washed with distilled water until waster become 37 clear. The samples were dried at 50 •C for 24 h. The isolated cellulose was stored in flask 38 and shelter from the light. The steps for cellulose extraction are represented in Fig. 1 The color of the samples was determined in a digital colorimeter model Chroma Me-46 ter CR-700 (Konica Minolta, Osaka, Japan), using the CIELab scale to determine L*, a* and 47 b* color parameters [13]. The sample was poured in a Petri dish with 5 cm diameter cov-48 ering the entire bottom of the dish, and the reading was performed in 10 different points. 49

Cellulose extraction
The total color difference (ΔE*) was calculated in relation to fresh vine stalks, according 1 to Eq. (1).
In this study, L*0, a*0 and b*0 stand for the values of the color parameters of fresh 4 stalk vine and L*, a* and b* for the values of the color parameters of the sample in each 5 step.  11 The samples were analyzed on a PerkinElmer Paragon 1000 FTIR (Waltham. MA. 12 USA) with an ATR accessory (Diamond/ZnSe). Spectra were obtained in the wavenumber 13 range of 4000-550 cm −-1 . with a resolution of 4 cm −-1 . accumulating 16 scans. Based on the 14 literature, the FTIR-ATR vibrational bands were identified [14]. 15

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During the extraction of cellulose from stalk vines, cellulose extracted from granulo-17 metric fractions with different particle sizes (500, 300, 250, and 150 µm and retain (<150)) 18 was characterized (Figure 1), to determine whether particle size influences cellulose ex-19 traction or the type of cellulose extracted. It was found that the observation with the Dino-20 Lite microscope was extremely important, both in the evaluation of the state of conserva-21 tion and in the measurement of sizes. Fig. 2 shows the photographs of the grape stalk fiber 22 treated with an autohydrolysis (acid, alkali and bleached) at 500 μm (a), 300 μm (b), 250 23 μm (c), 150 μm (d) and retain(e). All grape stalk fibers in the different diameters presented 24 opaque tint, after undergoing all treatments, presenting yellowish-brown coloration (Fig. 25  2). The white color desired during the process was not observed in the final product, in 26 order to be able to reach an indication of high purity of the cellulosic material. Thus, in 27 this method used, another hydrogen peroxide step is required for color change and 28 removal some traces of lignin and hemicellulose. Figure 2 depicts the topographic charac-1 terization of cellulose using a high-resolution digital microscope.  Table 1 displays the length of the vine stalks as well as the yield of cellulose extracted 5 from granulometric fractions with different particle sizes. 6 As shown in Figure 3, the bleaching procedure of cellulose obtained from different 7 fractions was also assessed using a colorimetric analysis using the CIELab scale as well as 8 the determination of the total color difference (ΔE*) in reference to fresh vine stalks to 9 further characterize the cellulose and analyze the influence of the particle size. Also, 10 shown in figure 3(d) a represented color from the CIELab values, from the stalk vine and 11 the bleached cellulose. Noticeably, based on figure 3(d) the bleaching process was not suf-12 ficient, and more cycles of hydrogen peroxide is needed. 13 Table 1. Yield of cellulose extracted from vine stalks with different particle size and length.  Figure 3. Analyses of color from colorimetry from different particle sizes of 500 µm, 300 µm, 250 µm, 19 150 µm and retain. The colorimetry was assed with CIELab and was evaluated the color from the 20 raw material (stalk vines) (a) and from the cellulose extracted after bleaching (b  Cellulose fractions, from the vines stalks, obtained from the production of wine treat-5 ment including bleaching processes were characterized in terms of functional groups us-6 ing FT-IR (Fig. 4). The spectra showed characteristic bands of the functional groups that 7 make up cellulose 2900, 1500 and 1200 cm -1 . According to the literature, it is confirmed 8 that bleaching indicated the partial removal of lignin, leaving some traces, due to the yel-9 lowish-brown color, requiring a later stage of bleaching to obtain a final solid fraction of 10 cellulose. Comparing the data obtained with the literature with cellulose from sugarcane 11 bagasse where they obtained an increase in the cellulose content in the final solid fraction, 12 using hydrogen peroxide at 35 % [15]. 13

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In this work was demonstrated the extraction of cellulose from stalk vines using a 15 modified auto-hydrolysis method. The use of vine stalks with different particle size al-16 lowed us to better understand the influence that the size as in the obtention of cellulose. 17 The observation with the Dino-Lite microscope and binocular magnifying glass illus-18 trated in Figure 2 was found to be particularly important in determining the level of con-19 servation as well as measuring sizes. The morphological structure of the micro cellulose 20 extracted appear to be long fibers in bigger particles such as 500, 300 and 250 µm . How-21 ever, particles smaller than 250 µm appear to have two distinctive shapes, rod-like and 22 long fibers. 23 Essentially, particles with larger particle sizes yielded more cellulose extracted. As 24 demonstrated in table 1, 500 µm particles produced cellulose yields of about 22 %, while 25 particles of less than 150 µm produced cellulose yields of only 3.1 %. 26 The fractionation process of cellulose, hemicellulose and lignin from stalk vine based 27 on the colorimetry and FTIR appeared to be insufficient. The fibers after bleaching shown 28 a yellow color, the author Vallejo et al., 2021 that also studied the extraction of cellulose 29 from the some agro-industrial residue obtained a final cellulose with a yellow under tone. 30 Even though the extraction method was different, it was generally challenging to separate 31 out well-delignified cellulose fractions from this residue. 32 It is also crucial to note that the way lignin molecules are arranged within the cell 33 walls of raw materials, such as their associations with other cell wall determines the effec-34 tive of the extraction process. Accordingly, the bleaching conditions must be optimized 35 and tailored to the characteristics of the agro-industrial residue if color and cellulose pu-36 rity are significant characteristics [2]. 37 Based on the results obtained on FTIR (figure 4) and the color (figure 3) the yellow 38 obtained was in fact residual lignin. Also, interment particles (250 and 150 µm ) had the 39 highest ΔE value indicating that the bleaching process was more effective. 40 It is expected that future research will yield an optimized method for extracting cel-41 lulose with a whiter color. Additionally, the optimized cellulose will be used to create 42 membranes/coatings for the protection of heritage buildings. The cellulose coatings will 43 be designed to shield buildings from external conditions such as heat. Funding: Adriana R. Machado thanks their research contract funded by Fundação para a Ciência e 1 Tecnologia (FCT) and project CENTRO-04-3559-FSE-000095 -Centro Portugal Regional Operational 2 Programe (Centro2020), under the PORTUGAL 2020 Partnership Agreement, through the European 3 Regional Development Fund (ERDF). The authors acknowledge the financial help from project 4 HAC4CG-Heritage, Art, Creation for Climate change. Living the city: catalyzing spaces for learn-5 ing, creation, and action towards climate change. NORTE-45-2020-75. SISTEMA DE APOIO À